Toxicity of hydroxylated polychlorinated biphenyls (HO-PCBs) using the bioluminescent assay Microtox<Superscript>®</Superscript>

Hydroxylated polychlorinated biphenyls (HO-PCBs) are toxic contaminants which are produced in the environment by biological or abiotic oxidation of PCBs. The toxicity of a suite of 23 mono-hydroxylated derivatives of PCBs and 12 parent PCBs was determined using the bacterial bioluminescent assay Microtox^®. All HO-PCBs tested exhibited higher toxicity than the corresponding parent PCB, with effect concentration 50 % (EC₅₀) ranging from 0.07 to 133 mg L^?1. The highest toxicities were recorded with 4-hydroxylated derivatives of di-chlorinated biphenyls (EC₅₀ = 0.07–0.36 mg L^?1) and 2-hydroxylated derivatives of tri-chlorinated biphenyls carrying a chlorine substituent on the phenolic ring (EC₅₀ = 0.34–0.48 mg L^?1). The toxicity of HO-PCBs generally decreased when the degree of chlorination increased. Consistently with this observation, a significant positive correlation was measured between toxicity (measured by EC₅₀) and octanol–water partition coefficient (pK _ow) for the HO-PCBs under study (Pearson’s correlation coefficient, r = 0.74), which may be explained by the lower solubility and bioavailability generally associated with higher hydrophobicity. This study is the first one which assessed the toxicity of a suite of PCBs and HO-PCBs using the bioluminescent assay Microtox^®, showing an inverse correlation between toxicity and hydrophobicity.     

Electronic data capture using the Womac<Superscript>®</Superscript> NRS 3.1 Index (m-Womac<Superscript>®</Superscript>): a pilot study of repeated independent remote data capture in OA

Aim

A preliminary evaluation of mobile phone technology for repeated independent remote data capture using the mobile phone-based m-WOMAC^® NRS 3.1 Index.

Methods

Following orientation to the m-WOMAC^® Index, and initial completion in the office, patients took the phones home and independently completed the Index on four subsequent occasions over 12 days, sending their data each time to a server in USA.

Results

Three men and nine women with hip (n = 2) and knee (n = 10) OA successfully completed the m-WOMAC^® Index on each occasion. Average time to completing the Index at termination was 4.8 min. The majority of patients rated logging on/opening the application, completing the m-WOMAC^® Index on the phone, and sending data as very easy (10–11/12), and were very confident (11/12) in continuing to use the phone to report their symptoms.

Conclusions

These data support the feasibility of repeated independent remote data capture using the m-WOMAC^® NRS3.1 Index.

     

Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript> and c[Nphe<Superscript>1</Superscript>,Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript>

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys^10,14]N/OFQ(1–14)NH₂ (c[Cys^10,14]) and its [Nphe¹] derivative c[Nphe¹,Cys^10,14]N/OFQ(1–14)NH₂ (c[Nphe¹,Cys^10,14]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation.In competition binding studies of rat, mouse and human NOP the following rank order pK_i was obtained: N/OFQ(1–13)NH₂(reference agonist)>N/OFQ=c[Cys^10,14]>>c[Nphe¹Cys^10,14]. In GTP³⁵S studies of Chinese hamster ovary cells expressing human NOP (CHO_hNOP) c[Cys^10,14] (pEC₅₀ 8.29) and N/OFQ(1–13)NH₂ (pEC₅₀ 8.57) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. Following 30 min pre-incubation c[Nphe¹Cys^10,14] competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pA₂ and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys^10,14] (pEC₅₀ 9.29, E_max 102% inhibition of the forskolin stimulated response), N/OFQ(1–13)NH₂ (pEC₅₀ 10.16, E_max 103% inhibition) and c[Nphe¹Cys^10,14] (~80% inhibition at 10 M) displayed agonist activity. In the mouse vas deferens c[Cys^10,14] (pEC₅₀ 6.82, E_max 89% inhibition of electrically evoked contractions) and N/OFQ(1–13)NH₂ (pEC₅₀ 7.47, E_max 93% inhibition) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. c[Nphe¹Cys^10,14] (10 M) competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pK_B of 5.66. In a crude attempt to assess metabolic stability, c[Cys^10,14] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH₂).In summary, we report that c[Cys^10,14] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe¹Cys^10,14] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTP³⁵S assay and agonist in cAMP assay) dependent activity.Presented in part to The British Pharmacological Society at the Brighton, UK Meeting January 2003     

HTLV-1 Infection and Neuropathogenesis in the Context of Rag1<Superscript>-/-</Superscript>γc<Superscript>-/-</Superscript> (RAG1-Hu) and BLT Mice

To date, the lack of a suitable small animal model has hindered our understanding of Human T-cell lymphotropic virus (HTLV)-1 chronic infection and associated neuropathogenesis defined as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Balb/c-Rag1^?/?γc^?/? or Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34⁺ hematopoietic stem cells. Flow cytometry and histological analyses confirmed reconstitution of Rag1 and BLT mice with human immune cells. Following HTLV-1 infection, proviral load (PVL) was detected in the blood of Rag-1 and BLT hu-mice as early as 2 weeks post-infection (wpi) with sustained elevation in the subsequent weeks followed by Tax expression. Additionally, infection was compared between adult and neonatal Rag1 mice with both PVL and Tax expression considerably higher in the adult Rag1 mice as compared to the neonates. Establishment of peripheral infection led to lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption within the central nervous system of infected mice. In addition, up-regulation in the expression of several immune checkpoint mediators such as programmed cell death-1 (PD-1), T-cell Ig and ITIM domain (TIGIT), and T cell Ig and mucin domain-3 protein (Tim-3) were observed on CD8⁺ T cells in various organs including the CNS of infected hu-mice. Collectively, these studies represent the first attempt to establish HTLV-1 neuropathogenesis in the context of Rag-1 and BLT hu-mice as potential novel tools for understanding HTLV-1 neuropathogenesis and testing of novel therapies such as immune checkpoint blockade in the amelioration of chronic HTLV-1 infection.     

The protective potential of <Emphasis Type="Italic">Yucca schidigera</Emphasis> (Sarsaponin 30<Superscript>®</Superscript>) against nitrite-induced oxidative stress in rats

The present study was designed to determine the protective effects of Yucca schidigera (Ys) against oxidative damage induced by acute nitrite intoxication as well as the histopathological evaluation of Ys in rats. The rats were divided into three groups each containing 12 rats: control (C); nitrite intoxication (N); Ys + nitrite intoxication (NY). C and N groups were fed standard rat feed (SRF). The NY group was fed SRF + 100 ppm Ys powder for 4 weeks. Acute nitrite intoxication was induced by subcutaneous (s.c.) administration of sodium nitrite (60 mg/kg) 1 day after the feeding period. Fifty minutes after sodium nitrite administration, blood samples and tissues including lung, liver, and kidney were collected for clinical biochemistry and histopathological investigations. Ys treatment was found to decrease methemoglobin, blood and tissue malondialdehyde, and tissue nitric oxide concentrations, and to increase the glutathione in blood and various tissues. However, plasma nitric oxide, total antioxidant activity, β-carotene, and vitamin A did not differ between N and NY groups. While the N group rats showed distinct pathology in various tissues (compared with controls), the NY group had similar lung and liver pathology to the control. Only moderate or mild hemorrhage and hyperemia were seen in kidneys of NY group rats. Consequently, the natural compounds found in Ys, such as polyphenols, steroidal saponins, and other phytonutrients, could be used to substantially protect the organism from nitrite-induced oxidative damage and its complications.     

Acute toxicity of Headline<Superscript>®</Superscript> fungicide to Blanchard’s cricket frogs (<Emphasis Type="Italic">Acris blanchardi</Emphasis>)

Previous laboratory studies have suggested that pyraclostrobin-containing fungicide formulations are toxic to amphibians at environmentally relevant concentrations. However, it is unknown if all pyraclostrobin formulations have similar toxicity and if toxicity occurs in different amphibian species. We investigated the acute toxicity of two formulations, Headline^® fungicide and Headline AMP^® fungicide, to Blanchard’s cricket frogs (Acris blanchardi) based on a direct overspray scenario. In addition, we examined body residues of fungicide active ingredients in A. blanchardi following direct exposure to Headline AMP fungicide. Headline fungicide and Headline AMP fungicide had similar toxicity to A. blanchardi with calculated median lethal doses of 2.1 and 1.7 µg pyraclostrobin/cm², respectively, which are similar to the suggested maximum label rate in North American corn (2.2 and 1.52 µg pyraclostrobin/cm², respectively). Tissue concentrations of pyraclostrobin were lower than predicted based on full uptake of a direct dose, and did not drop during the first 24 h after exposure. Headline fungicides at corn application rates are acutely toxic to cricket frogs, but acute toxicity in the field will depend on worst-case exposure.     

UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPγ<Superscript>35</Superscript>S binding studies

Studies of the pharmacology of nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) have been hampered by the lack of a range of high potency antagonists. In this study we have examined the effects of a novel N/OFQ analogue [Nphe(1),Arg(14),Lys(15)]N/OFQ NH(2) hereafter referred to as UFP-101. [(3)H]N/OFQ competition binding and GTPgamma(35)S binding assays were performed using CHO cells expressing the human NOP receptor (CHO(hNOP)). UFP-101 (pK(i) of 10.14+/-0.09) and a range of NOP selective agonists displaced [(3)H]N/OFQ binding with the following rank order of affinity: [Arg(14),Lys(15)]N/OFQ>[( pF)Phe(4)]N/OFQ(1-13)NH(2)>N/OFQ(1-13)NH(2)>UFP-101>N/OFQ>Ro64-6198>[Nphe(1)]N/OFQ(1-13)NH(2). N/OFQ, N/OFQ(1-13)NH(2), [( pF)Phe(4)]N/OFQ(1-13)NH(2), [Arg(14),Lys(15)]N/OFQ and Ro64-6198 also produced a concentration dependent (pEC(50) values of 8.75+/-0.11, 9.28+/-0.15, 9.69+/-0.04, 9.12+/-0.11 and 8.09+/-0.07 respectively) and saturable stimulation of GTPgamma(35)S binding and all were full agonists. UFP-101 did not stimulate GTPgamma(35)S binding per se, but produced a concentration dependent and parallel rightward shift in the concentration response curves to all agonists. UFP-101 yielded pA(2) values in the range 8.4-9.0. For comparison a pA(2) for [Nphe(1)]N/OFQ(1-13)NH(2) (the template for UFP-101) against N/OFQ of 7.33+/-0.08 was obtained. Slope factors for the Schild regression lines were approximately 1 indicating competitivity. When UFP-101 is compared with its template molecule [Nphe(1)]N/OFQ(1-13)NH(2), Arg(14),Lys(15) substitution produced approximately 1 log greater potency. We suggest that due to its high potency UFP-101 should prove a further useful tool in the evaluation of the N/OFQ-NOP receptor system.     

Constituents of PG201 (Layla<Superscript>®</Superscript>), a multi-component phytopharmaceutical,with inhibitory activity on LPS-induced nitric oxide and prostaglandin E<Subscript>2</Subscript> productions in macrophages

Fourteen compounds, coumarin (1), demethylsuberosin (2), xanthotoxin (3), psoralen (4), decursinol (5), decursin (6), decursinol angelate (7), chikusetsusaponin IVa (8), chikusetsusaponin IVa methyl ester (9), ethyl caffeate (10), syringaresinol (11), cnidilide (12), farnesol (13), and linoleic acid (14), were isolated from phytopharmaceutical PG201 (Layla^®) by activity-guided fractionation utilizing inhibitory activity on nitric oxide (NO) production in vitro. The isolates 1–14 were evaluated for their inhibitory activity on LPS-induced NO and prostaglandin E₂ (PGE₂) productions in RAW 264.7 cells. All the compounds except 14 displayed suppressive effects on LPS-induced NO and PGE₂ production with IC₅₀ values ranging from 8 to 60 μM. Among these, compound 10 showed the most potent inhibitory effect on NO production from RAW 264.7 cells with an IC₅₀ value of 8.25 μM. Compounds 2, 9, and 10 exhibited high inhibitory effects on PGE₂ production with the IC₅₀ values of 9.42, 7.51, and 6.49 μM, respectively. These findings suggest that compounds 2, 9, and 10 are the potential anti-inflammatory active constituents of PG201 and further study may be needed to explain their mechanism of action.     

Cannabinoid agonists differentially substitute for the discriminative stimulus effects of Δ<Superscript>9</Superscript>-tetrahydrocannabinol in C57BL/6J mice

RATIONALE: A variety of behavioral procedures have been developed to assess cannabinoid activity in mice; however, the feasibility of establishing Delta(9)-THC as a discriminative stimulus in mice has not been documented. OBJECTIVE: One goal was to establish Delta(9)-THC as a discriminative stimulus in mice; after having done so, another goal was to examine the in vivo mechanism of action of Delta(9)-THC with other cannabinoids and noncannabinoids. MATERIALS AND METHODS: C57BL/6J mice (n = 8) were trained to discriminate Delta(9)-THC (10 mg/kg i.p.) from vehicle while responding under a fixed ratio 30 schedule of food presentation. RESULTS: Mice satisfied the discrimination criteria in 18-98 (median = 67) sessions and the discriminative stimulus effects of Delta(9)-THC were dose-dependent (ED(50) = 2.6 mg/kg). CP 55940 and WIN 55212-2 dose-dependently increased Delta(9)-THC-appropriate responding to 100% (ED(50) = 0.032 and 0.45 mg/kg, respectively), whereas methanandamide and a variety of noncannabinoids (cocaine, ethanol, and ketamine) produced a maximum of 34% Delta(9)-THC-appropriate responding. The cannabinoid CB(1) antagonist SR 141716A (rimonabant) surmountably antagonized the discriminative effects of Delta(9)-THC, CP 55940, and WIN 55212-2; methanandamide did not significantly modify the Delta(9)-THC discriminative stimulus. CONCLUSIONS: The discriminative stimulus effects of Delta(9)-THC, CP 55940, and WIN 55212-2 are mediated by the same (i.e., CB(1)) receptors, whereas the effects of methanandamide or a metabolite of methanandamide are mediated at least in part by non-CB(1) receptors. The discriminative stimulus effects of Delta(9)-THC in mice could be used to evaluate mechanisms of cannabinoid activity with approaches (e.g., inducible knockouts) currently unavailable in nonmurine species.     

Pharmacologic Studies of a Prodrug of Mitomycin C in Pegylated Liposomes (Promitil<Superscript>®</Superscript>): High Stability in Plasma and Rapid Thiolytic Prodrug Activation in Tissues

Purpose

Pegylated liposomal (PL) mitomycin C lipid-based prodrug (MLP) has recently entered clinical testing. We studied here the preclinical pharmacology of PL-MLP.

Methods

The stability, pharmacokinetics, biodistribution, and other pharmacologic parameters of PL-MLP were examined. Thiolytic cleavage of MLP and release of active mitomycin C (MMC) were studied using dithiothreitol (DTT), and by incubation with tissue homogenates.

Results

MLP was incorporated in the bilayer at 10% molar ratio with nearly 100% entrapment efficiency, resulting in a formulation with high plasma stability. In vitro, DTT induced cleavage of MLP with predictable kinetics, generating MMC and enhancing pharmacological activity. A long circulation half-life of MLP (10–15 h) was observed in rodents and minipigs. Free MMC is either extremely low or undetectable in plasma. However, urine from PL-MLP injected rats revealed delayed but significant excretion of MMC indicating in vivo activation of MLP. Studies in mice injected with H3-cholesterol radiolabeled PL-MLP demonstrated relatively greater tissue levels of H3-cholesterol than MLP. MLP levels were highest in tumor and spleen, and very low or undetectable in liver and lung. Rapid cleavage of MLP in various tissues, particularly in liver, was shown in ex-vivo experiments of PL-MLP with tissue homogenates. PL-MLP was less toxic in vivo than equivalent doses of MMC. Therapeutic studies in C26 mouse tumor models demonstrated dose-dependent improved efficacy of PL-MLP over MMC.

Conclusions

Thiolytic activation of PL-MLP occurs in tissues but not in plasma. Liposomal delivery of MLP confers a favorable pharmacological profile and greater therapeutic index than MMC.

     

In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys<Superscript>7,10</Superscript>]N/OFQ(1–13)NH<Subscript>2</Subscript>

Nociceptin/orphanin FQ (N/OFQ) is the endogenous 17 amino acid peptide ligand for the G_i-protein-coupled N/OFQ receptor (NOP). In an attempt to improve the metabolic stability of N/OFQ, we have produced a truncated cyclic analogue with cysteine residues at positions 7 and 10, c[Cys^7,10]N/OFQ(1–13)NH₂ (c[Cys^7,10]). c[Cys^7,10], the template N/OFQ(1–13)NH₂ and N/OFQ displaced the binding of [³H]N/OFQ to Chinese hamster ovary cells expressing recombinant human NOP (CHO_hNOP) with pK _i values of 9.98, 9.83 and 9.18, respectively. In addition, c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ stimulated the binding of guanosine triphosphate gamma [³⁵S] to CHO_hNOP cells with pEC₅₀/E _max (stimulation factor) of 9.16/5.5, 9.11/4.9 and 8.35/5.5, respectively. c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation with pEC₅₀ values of 10.08, 10.11 and 9.78, respectively. All ligands produced complete inhibition of cAMP formation. In both functional assays, c[Cys^7,10] was a full agonist. In a series of metabolism experiments, incubation of 1 nM c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ with a rat brain homogenate produced a time-dependent loss of peptide that was greatest for the native peptide N/OFQ. Amidation in N/OFQ(1–13)NH₂ produced some metabolic protection, but this was not significantly improved by further inclusion of c[Cys^7,10]. In summary, c[Cys^7,10] is a high-affinity, high-potency full agonist of the NOP receptor. However, we were unable to demonstrate clear metabolic protection.     

Locomotor activity and respiration rate of the ground beetle, <Emphasis Type="Italic">Pterostichus oblongopunctatus</Emphasis> (Coleoptera: Carabidae), exposed to elevated nickel concentration at different temperatures: novel application of Multispecies Freshwater Biomonitor<Superscript>®</Superscript>

The aim of the work was to study effects of suboptimal temperature on nickel toxicity under chronic exposure and to apply the Multispecies Freshwater Biomonitor^® (MFB^®) for the first time to soil organisms in order to link behavioral to physiological endpoints. Ground beetles Pterostichus oblongopunctatus were reared at 10 or 20°C on control or Ni-contaminated (2500 mg Ni kg^?1) food. After 64 days half of the Ni-exposed beetles were transferred to uncontaminated food (elimination phase). The remaining Ni-exposed beetles were left on the contaminated food to the end of the experiment (96 days). After completing the experiment, respiration rate and locomotor activity were measured in Ni-contaminated beetles (Ni), Ni-contaminated ones after elimination (E), and controls (C). Then, the beetles were analyzed for Ni body loads. The respiration rate, which was always measured at 20°C for all experimental groups, was highest in Ni beetles reared at 10°C and did not differ between groups C and E. Similarly, locomotor activity was highest in Ni beetles, and marginally significant temperature effect was found. The study indicated thus that exposure to elevated Ni concentrations increased the maintenance costs in P. oblongopunctatus and that rearing the beetles at suboptimal temperature increased the respiration rate even further. However, the effects observed in both respiration rate and locomotor activity were reversible after decontamination. The study demonstrated also the potential of MFB^® for assessing the behavior of soil-dwelling organism in environmental toxicology.     

The sigma<Subscript>1</Subscript> (σ<Subscript>1</Subscript>) receptor activation is a key step for the reactivation of cocaine conditioned place preference by drug priming

Rationale Cocaine-seeking behavior can be investigated in rodents using the conditioned place preference (CPP) paradigm, in which the drug-paired environment serves as a conditioned stimulus. Such approach allowed to previously demonstrate the importance of the neuromodulatory sigma₁ (₁) receptor in acquisition of cocaine-induced CPP. CPP can be extinguished and then reactivated, notably using a cocaine challenge (i.e., priming).Objectives and methods In order to examine the role of the ₁ receptor in reinstatement of Cocaine-seeking, Swiss mice acquired CPP with cocaine (30 mg/kg, ip) and then CPP was extinguished.Results A challenge cocaine priming (15 mg/kg) reactivated CPP up to 140% of the post-conditioning response. Pre-administration of the ₁ receptor antagonist BD1047 (330 mg/kg, ip) or repeated treatment with an antisense probe targeting the ₁ receptor prevented CPP reactivation. The ₁ agonist igmesine (1–10 mg/kg, ip) or the steroid dehydroepiandrosterone (DHEA, 10–40 mg/kg, sc) reactivated CPP, in a BD1047-sensitive manner. Moreover, the in vivo [³H](+)-SKF-10,047 binding levels to the ₁ receptor were increased after cocaine conditioning in numerous brain structures and these increases subsisted after extinction. Finally, cross-reactivation of cocaine-induced CPP was observed after phencyclidine (PCP), morphine, nicotine and ethanol administration. However, BD1047 blocked reactivation of CPP induced by PCP, morphine and nicotine but not ethanol.Conclusions Since activation of the ₁ receptor is not sufficient to sustain CPP in naive animals [Neuropsychopharmacology 26 (2002) 444], it is concluded that ₁ receptor activation is a key event for relapse to drug seeking. Activation may occur via sensitization due to enhanced in vivo available of receptors.     

Cocaine and heroin (‘speedball’) self-administration: the involvement of nucleus accumbens dopamine and <Emphasis Type="Italic">μ</Emphasis>-opiate,but not <Emphasis Type="Italic">δ</Emphasis>-opiate receptors

Rationale The combined administration of heroin and cocaine (speedball) is common among intravenous drug users. Dopamine receptors in the nucleus accumbens play a key role in cocaine self-administration; however, their role in speedball self-administration is unknown, as is the role of opiate receptors in this region.Objectives The effect of blocking dopamine D1, D2, -opiate or -opiate receptors in the nucleus accumbens on the intravenous self-administration of combined heroin and cocaine was examined in rats.Methods Rats with bilateral cannulae implanted into the nucleus accumbens were trained to self-administer intravenous speedball (ratio of cocaine/heroin, 17:1) under a progressive ratio (PR) schedule. Prior to their self-administration session, rats were then microinjected with the dopamine D1 receptor antagonist SCH 23390 (1 and 6 nmol side^–1), the D2 receptor antagonist raclopride (3 and 10 nmol side^–1), the -opiate receptor antagonist CTOP (0.1, 0.3 and 1.0 nmol side^–1), the -opiate receptor antagonist naltrindole (1.0, 3.0 and 10 nmol side^–1) or a cocktail of SCH 23390 (1 nmol side^–1) and CTOP (0.1 nmol side^–1) into the nucleus accumbens.Results Microinjection of SCH 23390, raclopride or CTOP into the nucleus accumbens produced dose-dependent decreases in breakpoints under the PR schedule, while naltrindole was without effect. The highest dose of SCH 23390 also significantly reduced locomotor activity measured during speedball self-administration. The combination of SCH 23390 and CTOP significantly reduced breakpoints, while not affecting locomotor activity.Conclusions These results indicate that dopamine and -opiate receptors, but not -opiate receptors, in the nucleus accumbens are involved in the reinforcing effects of speedball. Combined administration of D1 and -opiate receptor antagonists may be more selective at reducing the reinforcing effects of speedball self-administration than either drug alone.     

Active cyamemazine metabolites in patients treated with cyamemazine (Tercian<Emphasis Type="Bold">®</Emphasis>): influence on cerebral dopamine D<Subscript>2</Subscript> and serotonin 5-HT<Subscript>2A</Subscript> receptor occupancy as measured by positron emission tomography (PET)

Rationale

Cyamemazine (Tercian®) is an antipsychotic agent blocking central dopamine D₂ receptors, which induces few extrapyramidal adverse effects, due to a potent antagonistic action at serotonin 5-HT_2A receptors. In vitro studies showed that the desmethyl metabolite of cyamemazine (N-desmethyl cyamemazine) has similar affinity for 5-HT_2A receptors as cyamemazine, whereas its D₂ receptor affinity is eight times lower (Benyamina et al. in Eur J Pharmacol 578(2–3):142–147, 2008). Moreover, cyamemazine sulfoxide showed modest affinity for 5-HT_2A receptors.

Objectives

The objective of this study is to measure steady-state plasma levels of N-desmethyl cyamemazine and cyamemazine sulfoxide in patients treated with clinically relevant doses of cyamemazine and correlate them with dopamine D₂ and serotonin 5-HT_2A receptor occupancies (RO) assessed by positron emission tomography (PET).

Methods

Eight patients received Tercian® 37.5, 75, 150, or 300 mg/day according to their symptoms. Dopamine D₂ and serotonin 5-HT_2A RO were assessed at steady-state cyamemazine plasma levels using [¹¹C]raclopride and [¹¹C]N-methyl-spiperone, respectively, for PET. Plasma levels of cyamemazine metabolites were determined using a validated high-performance liquid chromatography (PerkinElmer) associated with a mass spectrometry detection (API 365, PE SCIEX). The apparent equilibrium inhibition constant (K _i) was estimated by fitting RO with plasma levels of cyamemazine metabolites at the time of the PET scan.

Results

After 6 days of cyamemazine administration, plasma N-desmethyl cyamemazine reached steady-state levels at 2 to 12 times higher than those previously found for cyamemazine (Hode et al. in Psychopharmacology (Berl) 180:377–384, 2005). Plasma levels of N-desmethyl cyamemazine were closely related to striatal D₂ RO (r ²?=?0.942) and extrastriatal 5-HT_2A RO (r ²?=?0.901). The estimated K _i(app) value of N-desmethyl cyamemazine for striatal D₂ receptors was about fivefold higher than that for extrastriatal 5-HT_2A receptors (48.7 vs. 10.6 nM). Striatal D₂ RO increased with the plasma levels of N-desmethyl cyamemazine but remained below 75% even at its highest levels. At steady state, plasma cyamemazine sulfoxide levels were about double those of N-desmethyl cyamemazine. However, these cyamemazine sulfoxide levels should not contribute significantly to in vivo 5-HT_2A and D₂ receptor occupancy.

Conclusions

In patients orally given cyamemazine, N-desmethyl cyamemazine, but not cyamemazine sulfoxide, should significantly contribute to in vivo frontal 5-HT_2A and striatal D₂ receptor occupancy. The higher in vivo affinity of cyamemazine and its desmethyl metabolite for serotonin 5-HT_2A receptors compared with dopamine D₂ receptors should explain the low incidence of extrapyramidal adverse effects.

     

Intrinsic sympathomimetic activity of (-)-pindolol mediated through a (-)-propranolol-resistant site of the β<Subscript>1</Subscript>-adrenoceptor in human atrium and recombinant receptors

The -blocker (-)-pindolol produces intrinsic sympathomimetic activity manifested clinically by cardiostimulation, but the -adrenoceptor subtype, which mediates these effects, is unknown. Recent work indicates the existence of a (-)-propranolol-resistant site of the cardiac ₁-adrenoceptor and we propose that it mediates the cardiostimulation evoked by (-)-pindolol. We compared the interaction of (-)-pindolol both with human atrial myocardium and with recombinant ₁-adrenoceptors. The effects of (-)-pindolol on paced human atrial trabeculae were studied in the presence of 3-isobutyl-1-methylxanthine (IBMX; 20 M). (-)-Pindolol caused small negative and positive inotropic effects at nanomolar and micromolar concentrations respectively, which were unaffected by N^G-monomethyl-L-arginine (L-NMMA, 10 M), inconsistent with an involvement of nitric oxide. (-)-Pindolol, in the presence of (-)-propranolol, increased atrial contractile force and cAMP through recombinant ₁-adrenoceptors with identical potency (–logEC₅₀M=6.5). The positive inotropic effects of (-)-pindolol were resistant to blockade by L-748,337 (100 nM), a ₃-adrenoceptor antagonist. (-)-CGP12177, known to act through the (-)-propranolol-resistant site of the ₁-adrenoceptor, also increased with similar potency atrial contractile force (–logEC₅₀M=7.6) and cAMP at recombinant ₁-adrenoceptors (–logEC₅₀M=7.7). (-)-Pindolol blocked the effects of (-)-CGP12177 in human atrium and recombinant ₁-adrenoceptors with similar equilibrium dissociation constants (pK_B=6.5 and 6.3). Thus, stimulant potency and blocking potency of (-)-pindolol against (-)-CGP12177 agree. In contrast, (-)-pindolol was 200–400 times more effective at blocking the effects of a catecholamine than the effects of (-)-CGP12177 in both human atrium (pK_B=9.1) and at recombinant ₁-adrenoceptors (pK_B=8.6).We conclude that the cardiostimulant effects of (-)-pindolol in human atrial myocardium are mediated through a (-)-propranolol-resistant site of the ₁-adrenoceptor with low affinity for (-)-pindolol. In contrast, (-)-pindolol blocks the effects of catecholamines through a high-affinity site of the ₁-adrenoceptor. ₃-Adrenoceptors are not involved in the atrial effects of (-)-pindolol.     

Poly(ε-caprolactone)-<Emphasis Type="Italic">Block</Emphasis>-poly(ethyl Ethylene Phosphate) Micelles for Brain-Targeting Drug Delivery: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Valuation

Purpose  

The purpose of this work was to investigate the potential of poly(ε-caprolactone)-block-poly(ethyl ethylene phosphate) (PCL-PEEP) micelles for brain-targeting drug delivery.     

Studies on the reactions of 3,2′-polymethylene-2-phenylbenzo[<Emphasis Type="Italic">b</Emphasis>]-1,10-phenanthrolines with Ru(tpy)Cl<Subscript>3</Subscript> and properties of the products

A series of 3,2-polymethylene-2-phenylbenzo[b]-1,10-phenanthrolines was reacted with Ru(tpy)Cl₃ to afford two ruthenium (Ru) complexes, a pentaaza-coordinated (N5Cl) complex [Ru(tpy)(L)Cl]⁺ and a hexa-coordinated (N5C) complex [Ru(tpy)(L)]⁺. The ratio of these two complexes was found to be highly dependent on the length of the polymethylene bridge between terminal phenyl and central pyridine rings. The reaction between the dimethylene-bridged ligand and Ru(tpy)Cl₃ afforded a hexa-coordinated (N5C) complex as an only isolatable product in 83% yield, while the others afforded pentaaza-coordinated products and hexa-coordinated products in ratios of 1:1.6–3.5 with 80–90% overall yields. The UV spectra of pentaaza-coordinated complexes (N5Cl) and hexa-coordinated (N5C) cycloruthenated complexes were similar to show absorbances at 253–255, 276–286, 312–324, 360–373, and 490–532 nm. All of these complexes showed greenish blue light emissions in the range 450–460 nm upon excitation in the range 368–382 nm, while excitation at 500-532 nm resulted in green light emissions at 570 nm for pentaaza-coordinated complexes and 577–579 nm for hexaaza-coordinated species. Irradiation of the plasmid (100 μM) in the presence of 8c (λ_irr > 395 nm, 10 min) in air resulted in single-strand cleavage leading to the production of nicked plasmid (Form II), probably via intercalation.     

Effects of the serotonin transporter (5-HTTLPR) and α<Subscript>2A</Subscript>-adrenoceptor (C-1291G) genotypes on substance use in children and adolescents: a longitudinal study

Rationale and objective  

Twin studies suggest that substance use initiation in children and adolescents is determined primarily by environmental influences, whereas the establishment of use patterns is strongly controlled by genetic factors. The present study analysed the effects of the serotonin transporter promoter polymorphism [5-HT transporter gene-linked polymorphic region (5-HTTLPR)] and the α_2A-adrenoceptor C-1291G genotype (ADRA2A C-1291G) as well as their interaction effects on alcohol, tobacco and drug use from preadolescence to the late adolescence.     

Preferential effects of the cannabinoid CB<Subscript>1</Subscript> receptor antagonist,SR 141716, on food intake and body weight gain of obese (<Emphasis Type="Italic">fa/fa</Emphasis>) compared to lean Zucker rats

Rationale. The selective CB₁ receptor antagonist, SR 141716, has been demonstrated to reduce food consumption in a range of animal species. Objective. To assess the effect of chronic administration of SR 141716 on body weight and ingestive behaviour of lean and obese (fa/fa) Zucker rats. Methods. Lean and obese Zucker rats were orally dosed with SR 141716 (3, 10, 30 mg/kg PO), sibutramine (5 mg/kg PO) or vehicle for one week. Pair-fed controls provided insight as to whether the effect of SR 141716 on body weight was attributable to drug-induced hypophagia. Subsequently, the effect of chronic oral administration of SR 141716 (1, 3, 10 mg/kg) was assessed for 28 days. At the end of this period, all animals were given vehicle for 14 days. The incidence of wet-dog shakes, yawning, scratching, and grooming behaviours, was assessed after acute administration and at weekly intervals thereafter for 4 weeks. Results. SR 141716 dose-dependently decreased food intake and body weight gain in both lean and obese animals. The inhibition of food intake and body weight gain was greater in obese Zuckers than in lean Zucker controls. Changes in the body weights of pair-fed controls closely paralleled those of their drug-treated counterparts. Chronic 28-day treatment led to a maintained reduction of body weight gain. Withdrawal of SR 141716 on day 28 resulted in rebound hyperphagia and a significant weight gain. On acute administration, SR 141716 dose-dependently induced motor behaviours that showed tolerance upon repeated administration. Conclusion. These data indicate that chronic oral treatment with SR 141716 significantly reduces the food intake and body weight gain of obese and lean Zucker rats, an effect that is greater in obese animals and reversible upon drug withdrawal. Electronic Publication